Towards the Physical Map of the Trypanosoma cruzi Nuclear Genome: Construction of YAC and BAC Libraries of the Reference Clone T. cruzi CL-Brener

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Molecular Epidemiologic Typing Systems of Bacterial Pathogens: Current Issues and Perpectives

The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s )? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection). Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

Haemolymph and Fat Body Metallo-protease Associated with Enterobacter cloacae Infection in the Bloodsucking Insect, Rhodnius prolixus

Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus

Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Molecular epidemiology of methicillin resistant Staphylococcus aureus isolated from newborns in a hospital in Rio de Janeiro, Brazil

Methicillin resistant Staphylococcus aureus (MRSA) is an organism that is frequently transmitted in hospitals and perinatal units. The MRSA is considered a public health problem in neonatology because of its strong potential for dissemination in the wards associated with high rates of morbidity and mortality. In this study we describe the bacteriological, epidemiological and molecular characteristics of MRSA isolated from anterior nares and blood cultures of newborns hospitalized in a public maternity hospital in the city of Rio de Janeiro, Brazil. The frequency of MRSA isolated from nasal swabs of newborns was 47.8% (43/90). The genetic analysis of MRSA strains from anterior nares, showed 8 different pulsed field gel electrophoresis patterns (PFGE). Upon analysis of PFGE patterns of the 12 MRSA strains isolated from blood cultures, 8 different patterns were observed, 9 (75%) strains were genetic related to nasal secretion isolates patterns. In conclusion, our data demonstrate the importance of screening of newborns for the presence of MRSA in Brazilian hospitals and the usefulness of genetic typing of these pathogen during epidemiologic studies. This should lead to a better knowledge on the significancy and spreading of MRSA in the hospitals.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Genetic variability among populations of Lutzomyia (Psathyromyia) shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotominae) in Colombia

Polyacrylamide gel electrophoresis was used to elucidate genetic variation at 13 isozyme loci among forest populations of Lutzomyia shannoni from three widely separated locations in Colombia: Palambí (Nariño Department), Cimitarra (Santander Department) and Chinácota (Norte de Santander Department). These samples were compared with a laboratory colony originating from the Magdalena Valley in Central Colombia. The mean heterozygosity ranged from 16 to 22%, with 2.1 to 2.6 alleles detected per locus. Nei's genetic distances among populations were low, ranging from 0.011 to 0.049. The estimated number of migrants (Nm=3.8) based on Wright's F-Statistic, F ST, indicated low levels of gene flow among Lu. shannoni forest populations. This low level of migration indicates that the spread of stomatitis virus occurs via infected host, not by infected insect. In the colony sample of 79 individuals, the Gpi locus was homozygotic (0.62/0.62) in all females and heterozygotic (0.62/0.72) in all males. Although this phenomenon is probably a consequence of colonization, it indicates that Gpi is linked to a sex determining locus.

An outbreak of gastroenteritis associated with astrovirus serotype 1 in a day care center, in Rio de Janeiro, Brazil

Between June 4th and June 20th1996 rotavirus, adenovirus, and astrovirus (HAstrV) were investigated in fecal samples from 27 children under three years old with acute diarrhea, attending the Bertha Lutz day care center, in Rio de Janeiro. All fecal samples were analyzed by polyacrylamide gel electrophoresis (PAGE), reverse transcriptase polymerase chain reaction (RT-PCR), enzyme immunoassays (EIA), and electron microscopy (EM). Nine of them (33%) showed positive results for HAstrV by at least one of the employed methodologies. Eight were positive by RT-PCR and EIA, and six by EM. All positive samples were inoculated onto HT-29 (human colon adenocarcinoma) cultured cells for HAstrV isolation and seven were positive after three passages. The sequencing analysis of eight RT-PCR products (449 bp) from gene that codifies VP2 protein, showed a total nucleotide identity among them and 98% with HAstrV-1 (strain Oxford type 1). This is the first report of a gastroenteritis outbreak associated with HAstrv-1 in a day care center in Rio de Janeiro and it reinforces the importance of this virus in association with infantile acute gastroenteritis.

Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Epidemiological features of rotavirus infection in Goiânia, Goiás, Brazil, from 1986 to 2000

A total of 2,605 faecal specimens from children up to 10 years old with or without diarrhoea were collected. Samples were obtained from 1986 to 2000 in hospitals, outpatient clinics and day-care centers in Goiânia, Goiás. Two methodologies for viral detection were utilized: a combined enzyme immunoassay for rotavirus and adenovirus and polyacrylamide gel electrophoresis. Results showed 374 (14.4%) faecal specimens positive for Rotavirus A, most of them collected from hospitalized children. A significant detection rate of rotavirus during the period from April to August, dry season in Goiânia, and different frequencies of viral detection throughout the years of study were also observed. Rotavirus was significantly related to hospitalization and to diarrhoeal illness in children up to 24 months old. This study reinforces the importance of rotavirus as a cause of diarrhoea in children and may be important in regards to the implementation of rotavirus vaccination strategies in our country.